Clostridium botulinum produces seven different neurotoxins (BoNTs) which differentiated serologically by the lack of anti-serum cross serotype neutralization. BoNTs are the most potent toxins known to humans and are the causative agents of the disease botulism (1). BoNTs exert their action by inhibiting the release of the neurotransmitter acetylcholine at the neuromuscular junction, leading to a state of flaccid paralysis. BoNTs elicit neuronal-specific flaccid paralysis by targeting neurons and cleaving neuron specific SNARE proteins.
SNARE proteins (Soluble NSF Attachment protein Receptors) are a large superfamily of proteins. The main function of SNARE proteins is to mediate the exocytosis of neurotransmitter molecules to the post-synaptic junction. SNAREs are small, abundant and both vesicle and plasma-membrane bound proteins.
BoNTs are a 150 kDa polypeptide chain comprising a 100 kDa heavy chain and a 50 kDa light chain linked by a disulfide bond. BoNTs are organized into three functional domains: an N-terminal zinc metalloprotease light chain (LC), a translocation domain (HCT) and a C-terminal receptor binding domain (HCR) (1, 2). The toxic effect of BoNTs (nerve intoxification) is accomplished through the interplay of three key events. One, the carboxy half of the heavy chain is required for receptor-specific binding to cholinergic nerve cells at the nerve-cell membrane. After binding, another portion of the BoNT moves a smaller catalytic domain into the cell, where the catalytic domain binds to and cleaves a neuronal SNARE protein, “intoxicating” the nerve cell, making it impossible to “fire” or send signals. By “catalytic domain” we mean the part of the molecule that triggers the cleavage of the substrate. The toxin is internalized into an endosome through receptor-mediated endocytosis, and the toxin binds the liminal domains of synaptic vesicle-associated proteins upon the fusion of synaptic vesicles with the plasma membrane (3-5). In short, BoNTs are internalized into endosomes and upon acidification, the LC is translocated into the cytoplasm, where SNARE proteins are cleaved (1, 2).
Mammalian neuronal exocytosis is driven by the formation of protein complexes between the vesicle SNARE, VAMP2, and the plasma member and SNAREs, SNAP 25 and syntaxin 1a (6). There are seven serotypes of BoNTs (termed A-G) that cleave specific residues on one of three SNARE proteins: serotypes B, D, F, and G cleave VAMP-2, serotypes A and E cleave SNAP 25, and serotype C cleaves SNAP 25 and syntaxis 1a (1). Thus, neuronal specificity is based upon BoNT binding to neurons and cleaving neuronal isoforms of the SNARE proteins. For example, BoNT/A cleaves human SNAP25, but not the human non-neuronal isoform SNAP 23 (7, 8). The non-neuronal SNARE isoforms are involved in a divergent cellular processes, including fusion reactions in cell growth, membrane repair, cytokinesis and synaptic transmission.
The reversible nature of muscle function after BoNTs intoxication that replace toxin-affected nerves with new nerves (10, 11), has turned the BoNTs from a deadly agent to novel therapies for neuromuscular conditions. As early as 1989, BoNT/A was approved by the FDA to treat strabismus, blepharospasm, and hemifacial spasm and then for cervical dystonia, cosmetic use, glabellar facial lines and axillary hyperhidrosis (12). BoNT/A efficacy in dystonia and other disorders related to involuntary skeletal muscle activity, coupled with a satisfactory safety profile, and prompted empirical/off-label use in a variety of secretions and pain and cosmetic disorders (13).
The clinical use of BoNTs is limited to targeting inflictions affecting neuromuscular activity (12, 13). Elucidation of the structure-function relationship of BoNTs has enabled the design of novel therapies that retarget BoNT to unique neurons and non-neuronal cells. Replacement of BoNT HCR domain with nerve growth factor, lectin from Erythrina cristagalli, or epidermal growth factors enable retargeting of BoNT/A to neuronal or non-neuronal cells such as nociceptive afferents and airway epithelium cells (14-16). However, the selective cleavage of neuronal specific SNARE proteins by BoNT has limited development of novel therapies in these non-neuronal systems. Prerequisite to develop novel therapies requires the retargeting of the catalytic activity of the BoNTs to non-neuronal SNARE isoforms.
Accordingly, a need exists for an engineered BoNT that cleaves non-neuronal SNARE proteins and methods of use thereof.